The release of pro-inflammatory cytokines is a critical early event in host pathogen defense, often limiting the initial spread of infectious agents through innate mechanisms and activating the adaptive response for more long-term control or complete elimination of infection. Because these immunologic processes can be damaging to host tissue, the amount and time course of cytokine release must be finely controlled and numerous pathologies are associated with dysregulated cytokine activity. The molecular mechanisms used in fine-tuning this process are therefore the subject of active investigation. Here we propose using a forward genetic analysis of inbred (C57BL/6J) and wild derived (MOLF/Ei, Czech/EiII, MSM/Ms) mouse strains that secrete different amounts of IL-6 in response to TLR stimulation as a novel approach to characterizing the regulation of early cytokine release. Initial studies from a panel of backcross mice have identified two loci on chromosomes 6 and 9 that make major contributions to the trait. Further, these two loci have a significant epistatic interaction (p < 10-6, LRS 48.1). Our mapping indicates that these loci contain candidate genes not previously implicated in TLR-mediated signaling. We propose to map these genes via meiotic recombination (and test the hypothesis that allelic variation at these loci contributes to the phenotype?). We also propose to establish the mechanistic basic for their observed epistatic interaction. This proposal aims to approach the problem through more refined genetic analysis as well as biochemical and molecular characterization of the candidate genes. We believe that we will reveal important novel mechanisms of TLR signaling.